Bioprinting of human pluripotent stem cell derived corneal endothelial cells with hydrazone crosslinked hyaluronic acid bioink.

BACKGROUND: Human corneal endothelial cells lack regenerative capacity through cell division in vivo. Consequently, in the case of trauma or dystrophy, the only available treatment modality is corneal tissue or primary corneal endothelial cell transplantation from cadaveric donor which faces a high global shortage. Our ultimate goal is to use the state-of-the-art 3D-bioprint technology for automated production of human partial and full-thickness corneal tissues using human stem cells and functional bioinks. In this study, we explore the feasibility of bioprinting the corneal endothelium using human pluripotent stem cell derived corneal endothelial cells and hydrazone crosslinked hyaluronic acid bioink. METHODS: Corneal endothelial cells differentiated from human pluripotent stem cells were bioprinted using optimized hydrazone crosslinked hyaluronic acid based bioink. Before the bioprinting process, the biocompatibility of the bioink with cells was first analyzed with transplantation on ex vivo denuded rat and porcine corneas as well as on denuded human Descemet membrane. Subsequently, the bioprinting was proceeded and the viability of human pluripotent stem cell derived corneal endothelial cells were verified with live/dead stainings. Histological and immunofluorescence stainings involving ZO1, Na+/K+-ATPase and CD166 were used to confirm corneal endothelial cell phenotype in all experiments. Additionally, STEM121 marker was used to identify human cells from the ex vivo rat and porcine corneas. RESULTS: The bioink, modified for human pluripotent stem cell derived corneal endothelial cells successfully supported both the viability and printability of the cells. Following up to 10 days of ex vivo transplantations, STEM121 positive cells were confirmed on the Descemet membrane of rat and porcine cornea demonstrating the biocompatibility of the bioink. Furthermore, biocompatibility was validated on denuded human Descemet membrane showing corneal endothelial -like characteristics. Seven days post bioprinting, the corneal endothelial -like cells were viable and showed polygonal morphology with expression and native-like localization of ZO-1, Na+/K+-ATPase and CD166. However, mesenchymal-like cells were observed in certain areas of the cultures, spreading beneath the corneal endothelial-like cell layer. CONCLUSIONS: Our results demonstrate the successful printing of human pluripotent stem cell derived corneal endothelial cells using covalently crosslinked hyaluronic acid bioink. This approach not only holds promise for a corneal endothelium transplants but also presents potential applications in the broader mission of bioprinting the full-thickness human cornea.

Cornea-Specific Human Adipose Stem Cell-Derived Extracellular Matrix for Corneal Stroma Tissue Engineering.

Utilizing tissue-specific extracellular matrices (ECMs) is vital for replicating the composition of native tissues and developing biologically relevant biomaterials. Human- or animal-derived donor tissues and organs are the current gold standard for the source of these ECMs. To overcome the several limitations related to these ECM sources, including the highly limited availability of donor tissues, cell-derived ECM offers an alternative approach for engineering tissue-specific biomaterials, such as bioinks for three-dimensional (3D) bioprinting. 3D bioprinting is a state-of-the-art biofabrication technology that addresses the global need for donor tissues and organs. In fact, there is a vast global demand for human donor corneas that are used for treating corneal blindness, often resulting from damage in the corneal stromal microstructure. Human adipose tissue is one of the most abundant tissues and easy to access, and adipose tissue-derived stem cells (hASCs) are a highly advantageous cell type for tissue engineering. Furthermore, hASCs have already been studied in clinical trials for treating corneal stromal pathologies. In this study, a corneal stroma-specific ECM was engineered without the need for donor corneas by differentiating hASCs toward corneal stromal keratocytes (hASC-CSKs). Furthermore, this ECM was utilized as a component for corneal stroma-specific bioink where hASC-CSKs were printed to produce corneal stroma structures. This cost-effective approach combined with a clinically relevant cell type provides valuable information on developing more sustainable tissue-specific solutions and advances the field of corneal tissue engineering.